SOIL MANUAL

CONTENT
1. Soil sample registration & preparation in the laboratory for analysis

2. Determination of soil texture – international pipette method

(mechanical analysis)

3. Soil moisture

4. Determination of Soil pH and Electrical Conductivity

5. Cation exchange capacity

6. Organic Carbon

7. Mineralizable N

8. Inorganic Nitrogen

9. Ammonium by indophenol blue method

10. Available Phosphorus

11. Available Potassium

12. Available Sulphur

13. Micronutrients - available Zn, Cu, Fe, Mn, B and Mo

 1. SOIL SAMPLE REGISTRATION & PREPARATION IN THE LABORATORY
FOR ANALYSIS

Registration of Samples :
1. The soil samples are received in the Sample Receiving Cell, where the condition
and quantity of the samples are examined and acknowledgement slip is issued to the
person delivering the sample.
2. The entry of soil sample is taken in the Sample Register (office) as per the
relevant particulars furnished in the Register. Lab. No. is given to each sample.
3. Test required and expected date of reporting and other details along with the
sample is then transferred to analysis.

Soil Sample Preparation for Analysis :

1. The collected soil samples are homogeneously mixed and left to attain equilibrium
with air for 2 hours in the trays / paper dishes.
2. If the samples are dry there is no need to keep the samples in the oven. It can be
directly taken for further testing.
3. If the soil samples are wet, samples are dried in the oven at 25oC for 2 hours or
more (depending upon the wetness of the sample). If samples are found sticky even
after drying, then the temperature may be raised by 2 to 5oC, but in any case, it
should not exceed 35oC.
4. After drying the soil, clods are crushed gently and grounded with the help of
wooden pestle and mortar. Gravel, soft chalk, limestone, stones and concretions
should be removed from the samples.
5. The soils are passed through 2.0 mm and 0.5 mm stainless steel sieve. The
sufficient quantity of sieved soil sample is kept in plastic bag labeled with permanent
ink marker (IC No. along with the sieve size 2.0 mm / 0.5 mm).may be discarded.
6. The plant residues, gravel, and other materials retained on the sieve may be
discarded.
7. If the gravel content is substantial, the percent of the sample (W/W) may be
recorded.
8. After the analysis of samples, the results are reviewed by the Higher Authority and
entered in appropriate register.
9. The test reports should be signed by the authorized person.
Reviewing of Soil Samples :
1. After the completion of the analysis, the remaining samples are stored in the
sampling room.
2. The advisory soil samples having test results not in normal range are retained for
one month after sending the test results.
3. The samples are retained to recheck the test results, if any query is raised.
4. The soil samples of research studies are retained depending on the objective of
the investigation of the project.

Reviewing of Soil Samples :

1. After the completion of the analysis, the remaining samples are stored in the
sampling room.
2. The advisory soil samples having test results not in normal range are retained for
one month after sending the test results.
3. The samples are retained to recheck the test results, if any query is raised.
4. The soil samples of research studies are retained depending on the objective of
the investigation of the project
2. DETERMINATION OF SOIL TEXTURE – INTERNATIONAL PIPETTE METHOD
(MECHANICAL ANALYSIS)
Purpose :
The process of determining the amount of individual size separates of soil below 2
mm in diameter i.e. sand, silt and clay called particle size analysis. Particle size
distribution has an important influence on soil permeability or water intake rate, water
storage capacity ability to aggregate, crushing and the chemical makeup of the soil.
The value of land, land use capability and soil management practices are largely
determined by the texture. The size limits for different fractions according to
International system of classification is as follows – ISSS.
a) Gravel Greater than 2 mm diameter
b) Coarse Sand 2.0 to 0.2 mm diameter
c) Fine Sand 0.2 to 0.02 mm diameter
d) Silt 0.02 to 0.002 mm diameter
e) Clay Less than 0.002 mm diameter

The size limits for different fractions are also classified according to
Principle :
Mechanical analysis of soils consists of essentially two distinct operations.
i) Dispersion of the soil
ii) Determination of particle size distribution by sedimentation (by Stokes Law)

Apparatus :
i) Shaking machine
ii) 1000 ml measuring stoppered glass cylinder
iii) Thermometer
iv) Pipette
v) Stirrer
vi) Stop watch
vii) Oven
viii) Physical balance
ix) 500 ml plastic bottle.
x) Sieve (2, 0.2, and 0.02 mm)
xi) China dishes

Reagents :
i) Sodium hexa-meta phosphate [NaPO3]6
ii) Sodium carbonate (Na2CO3)
iii) Dispersing reagent - Take 33 gm of Sodium Hexametaphosphate and add 7 gm
of Sodium carbonate and make the volume 1 litre with distilled water.
Procedure :
i) For determination of soil texture, take 50 gm of air dry soil (passed through 2 mm
sieve) in 500 ml bottle.
ii) Add 100 ml above dispersion solution in 50 gm soil in 500 ml plastic bottle.
iii) Shake a set of sample bottles at regular intervals for half an hour on shaking
machine for preparing homogeneous solution.
iv) Transfer above soil sample solution transferred to 1000 ml glass measuring
cylinder and make solution 1000 ml by adding water.
v) As per International approved system, shake the sample solution for 30 sec.
Depending on the solution temperature and sedimentation chart, first pipetting is
done with 50 ml pipette at 10 cm depth. In first pipetting, 50 ml solution sucked and
transferred in 60 ml china dish. This sample solution contains mixed of clay and silt
particles.
vi) Depending on the solution temperature and sedimentation chart, second pipetting
is done with 50 ml pipette at 10 cm depth. In second pipetting 50 ml solution sucked
and
transferred in 60 ml china dish. This solution contains clay particles in soil sample.
vii) Transfer remaining soil solution in 1 lit. measuring cylinder by using 0.02 mm
sieve and wash the material through the sieve using jet of water. Sand particles on
sieve are collected in china dish.
viii) Transfer pipetted solution in 3 dishes and dry overnight in an oven at 105oC,
Cool in a desiccators and weigh quickly.
ix) The weight of fine sand determined by deducting the weight of clay, silt and
coarse sand particle from 100.
Calculations :
Coarse Sand (A gm) x 2 = ………..……..% Coarse sand

In 50 gm soil sample A gm Coarse sand

100 gm soil sample A gm x 2 …… Coarse sand (I)

In 1000 ml sample solution → 50 gm soil

In 50 ml sample solution → 2.5 gm soil

In 2.5 gm sample → B gm …… (Clay + Silt)

In 100 gm sample→ (B x 100) / 2.5 = B x 40 gm … (Clay + Silt)

In 2.5 gm sample → C gm …… (clay)

100 gm sample → C x 40 gm …… (clay) (II)

% Silt = (B x 40) – (C x 40) (III)

% Coarse sand = --------- (1)

% Clay = ----------- (2)

% Silt = ----------- (3)

% Fine sand = 100 – (1) + (2) + (3)

 3. Soil moisture
Direct method (Gravimetric method):
This is the simplest and most widely used method for measuring soil moisture.
Equipments:
a. Sampling tube/auger
b. Moisture cans (numbered)
c. Balance with weights/automatic
d. Drying oven
e. Desiccator
Procedure :
Collect soil samples by tube or auger from a number of points within the
experimental site and mix thoroughly. Place composite sub samples of about 50
gm to 100 gm in soil moisture cans with tight fitting lids. Take atleast three sub-
samples. The moist samples are weighed immediately, dried to constant weight in
an oven at 105°C (for about 24 hrs) and reweighed after cooling in a desiccator.
Determine the tare weight of moisture cans.
Calculate the soil moisture content by determining the loss in weight on drying
and the weight of the oven dry soil as follows:
wt. of wet soil + tare) − (wt. of dry soil + tare)
𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑏𝑏𝑏𝑏 𝑤𝑤𝑤𝑤. (%) =
(𝑤𝑤𝑤𝑤. 𝑜𝑜𝑜𝑜 𝑑𝑑𝑑𝑑𝑑𝑑 𝑠𝑠𝑠𝑠𝑠𝑠𝑙𝑙 + 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) − (𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡)
loss of wt. on drying
𝑀𝑀𝑀𝑀(%) = × 100
𝑤𝑤𝑤𝑤. 𝑜𝑜𝑜𝑜 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑑𝑑𝑑𝑑𝑑𝑑 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠
𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏
= 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑜𝑜𝑜𝑜 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤ℎ𝑡𝑡 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 × 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑
4. Determination of Soil pH and Electrical Conductivity
Determination of pH is actually a measurement of hydrogen ions activity in soil –
water system. It is defined as negative logarithm of the hydrogen ion activity.
Mathematically, it is expressed as:
pH = - log a [H+ ]
The pH value of a soil is an indication of soil reaction i.e. acidic, neutral or
alkaline. The nutrient availability is governed by soil reaction. It is maximum at
neutral pH and decreases with increase in acidity or alkalinity. Thus, pH value
gives an idea about the availability of nutrients to plants.
Principle:
The pH is usually measured by pH meter, in which the potential of hydrogen ion
indicating electrode (glass electrode) is measured potentiometrically against
calomel saturated reference electrode which also serves as salt bridge. Now a
days, most of the pH meters have single combined electrode. Before measuring
the pH of the soil, the instrument has to be calibrated with standard buffer solution
of known pH. Since, the pH is also affected by the temperature. Hence, the pH
meter should be adjusted to the temperature of the solution by temperature
correction knob.

Instrument:
pH meter
A pH meter is used to determine the acidity or alkalinity of the solution.
Hydrogen ions in solution, like other ionic species, conduct an electric current.
pH is the concentration of hydrogen ions in the solution. A solution containing
more H+ ions remains acidic while the solution containing more OH- ions
remains alkaline.
A pH meter is an electronic voltmeter that measures this difference of potential
and through its internal calibration, converts it to a pH reading which is
displayed on a scale. pH value of solutions ranges from 1 to 14. pH meter is
used to determine the pH of different solutions and is much better than pH
strips.
Nobel-Prize winning German chemist Fritz Haber (1868–1934) and his student
Zygmunt Klemensiewicz (1886–1963) developed the glass electrode idea in
1909. The modern, electronic pH meter was invented about a quarter century
later, in 1934, when American chemist Arnold Beckman (1900–2004) figured
 out how to hook up a glass electrode to an amplifier and voltmeter to make a
much more sensitive instrument. He was a granted a patent in October 1936.
Components:
• The glass pH probe contains two electrodes i.e. the glass electrode, has
a silver-based electrical wire suspended in a solution of potassium chloride,
contained inside a thin bulb (or membrane) made from a special glass containing
metal salts (typically compounds of sodium and calcium). The other electrode is
called the reference electrode and has a potassium chloride wire suspended in a
solution of potassium chloride.
• Temperature probe
• Electronic control unit
Working:
• Suppose the unknown solution is much more acidic, so it contains a lot more
hydrogen ions. What the glass electrode does is to measure the difference in pH
between the inside solution and the outside test solution by measuring the
difference in the voltages their hydrogen ions produce. Since we know, potassium
chloride inside the glass electrode is a neutral solution with a pH of 7, so it
contains a certain amount of hydrogen ions (H+), we can figure out the pH of the
outside solution.
• When you dip the two electrodes into the test solution, some of the hydrogen ions
move toward the outer surface of the glass electrode and replace some of the
metal ions inside it, while some of the metal ions move from the glass electrode
into the test solution. This ion-swapping process is called ion exchange, and it's
the key to how a glass electrode works.
• Ion-swapping also takes place on the inside surface of the glass electrode from
the inside solution. The two solutions on either side of the glass have different
acidity, so a different amount of ion-swapping takes place on the two sides of the
glass.
• This creates a different degree of hydrogen-ion activity on the two surfaces of the
glass, which means a different amount of electrical charge builds up on them. This
charge difference means a tiny voltage (sometimes called a potential difference,
typically a few tens or hundreds of millivolts) appears between the two sides of the
glass, which produces a difference in voltage between the silver electrode (5) and
the reference electrode (8) that shows up as a measurement on the meter.
• Although the meter is measuring voltage, what the pointer on the scale (or digital
display) actually shows us is a pH measurement.
• The bigger the difference in voltage between the inside and outside solutions, the
bigger the difference in hydrogen ion activity between.
• If there is more hydrogen ion activity in the outside solution, it's more acidic than
the inside solution and the meter shows this as a lower pH; in the same way, if
there's less hydrogen ion activity in the outside solution, the meter shows this as a
higher pH (more alkaline).

Precautions:
• Never touch the membrane of the glass electrode with anything else except
soft tissue paper since it is fragile and is easily ruined if scratched or bumped.
• The electrodes must not be removed from the solution unless the selector
switch is at zero.
• Never dip the glass electrode in a solution with a dehydrating action.
• If used for measuring pH of albuminous substances, the glass electrode must
be cleaned with suitable solvents and then the electrode be placed in distilled
water for a few hours before it is used to measure the pH of the other solution.
• For basic solutions with pH more than 11, gIass electrodes of special
composition are required to avoid interference due to sodium ion.
• The glass electrode may be covered with a sleeve to save it from jerks.

Reagents:
Standard buffer solutions: These may be of pH 4.0, 7.0 or 9.2 and are
prepared by dissolving one standard buffer tablet in 100 ml distilled water. It is
necessary to prepare fresh buffer solution after few days.
In absence of buffer tablet, a 0.05 M potassium hydrogen phthalate solution
can be used which gives a pH of 4.0 (Dissolve 10.21 g. of A.R. grade
potassium hydrogen phthalate in distilled water and dilute to 1 litre. Add 1 ml
of chloroform or a crystal of thymol per litre as a preservative).
Procedure:
(a) Soil to water ratio of 1:2 (pH2)
Take 20 g soil in 100 ml beaker and add 40 ml. of distilled water to it. The
suspension is stirred at a regular interval for 30 minutes. Determine the pH by
immersing electrodes in suspension. For soils containing high salts, the pH
should be determined by using 0.01M calcium chloride solution. (Dissolve
0.110 g of CaCl2 in water and dilute to 1 litre).

(b) Saturate soil paste (pHs)

Add small amount of distilled water to 250g of air dried soil. Stir the mixture
with a spatula. At saturation, the soil paste glistens and flows slightly when the
container is tapped it slides freely and ensures cleanly off the spatula. After
mixing, allow the sample to stand for an hour. If the paste has stiffened
markedly or lost its glistening, add more water or if free water has collected on
the surface of the paste, add an additional weighed quantity of dry soil and
mix it again. Then insert the electrode carefully in the paste and measure the
pH.
(c) Saturation extract (pHe)
The soil is extracted using vacuum extractor and the pH is measured in the
saturation extract.

Soil reaction ratings

pH range Soil reaction rate

<4.6 Extremely acidic

4.6–5.5 Strongly acidic
5.6-6.5 Moderately
acidic
6.6-6.9 Slightly acidic
7.0 Neutral
7.1-8.5 Moderately
alkaline
>8.5 Strongly alkaline
Conductivity:
The knowledge of total soluble salts is essential in crop production, especially
during the process of salinization. Since, there is a direct relationship between
the quantity of soluble salts and the electrical conductance. Hence soluble
salts in soils are measured indirectly by measuring the electrical conductance
of the soil.
Principle:
The electrical conductivity is measured with the help of solu bridge. The
instrument is calibrated and cell constant is determined with the help of 0.1 N KCl
solution. This solution gives an electrical conductance of 1.41 mmhos/cm or
dSm-1 at 250C.

Reagents:
Potassium chloride: Dissolve 0.7456g dry potassium chloride (AR) in distilled
water and make up the volume to one litre.

Procedure:
Take 20 g of soil in 100 ml beaker, add 40 ml of distilled water and shake
intermittently for 30 minutes. Determine the conductivity of the supernatant
liquid with the help of conductivity meter. The electrical conductivity of
saturation extract (E.C.e) is also determined for salinity ratings.

Chemical characteristics of saline, non-saline sodic and saline sodic soils

Soil EC Exchangeable pH
(dS/m) sodium
percentage
saline > 4.0 < 15 < 8.5
Sodic(non- < 4.0 > 15 > 8.5
saline)
Saline sodic > 4.0 > 15 < 8.5

General interpretation of EC values

Soil EC Total salt Crop reaction

(mS/cm) content
(%)
Salt free 0-2 < 0.15 Salinity effect
negligible,
except for more
 sensitive crops

Slightly saline 4-8 0.15–0.35 Yield of many

crops restricted
Moderately 8-15 0.35–0.65 Only tolerant
saline crops yield
satisfactorily
Highly saline >15 > 0.65 Only very
tolerant crops
yield
satisfactorily
 5. Cation exchange capacity
The total number of exchangeable cations a soil can hold is called its cation
exchange capacity (CEC). The higher the CEC, the more cations it can retain.
It can be expressed in terms of milli-equivalents per 100 g of soil (me/100 g)
or in centimoles of positive charge per kilogram of soil (cmol/kg), which is
numerically equal to me/100 g. The CEC of the soil depends on the kind of
clay and OM present.
Apparatus:
The apparatus required in order to determine the CEC consists of:
• a centrifuge;
• some 50-ml round-bottom centrifuge tubes;
• a mechanical shaker;
• a flame photometer and accessories that include propane, lithium and
sodium standards.
Instrument:
Flame photometer
Principle:
Flame photometry is the method used to determine the elements which can get
easily excited that are alkali metals (Li, Na, K etc) and alkaline earth metals (Ca,
Ba, Mg etc). This method is based upon the measurement of intensity of radiation
emitted, in the visible region, when a metal atom is introduced into a flame. The
wavelength of the radiation (or the colour), emitted tells us about the element, and
the intensity of the radiation tells us the concentration of the element present.
Working:
In flame photometry the sample is introduced into a flame wherein it undergoes a
number of processes leading to the formation of excited atomic species which
emit radiation. The radiation is then measured and analyzed. The instrument used
for the purpose is called flame photometer. It consists of the following basic
components:
• Flame atomiser: It converts the sample into excited atomic species and consists of
the following.
• Nebuliser and mixing chamber: It is a means of transporting a homogeneous
solution into the flame at a steady rate.
• Atomiser burner: The fuel and oxidant burn to give a flame that can be maintained
in a constant form and at a constant temperature.
• Monochromator (or filter): It isolates the light of the wavelength to be measured
from that of irrelevant emissions.
• Detector: It helps in measuring the intensity of radiation emitted by the flame.
• Amplifier and Readout Device: It is used to amplify the signal and provides a
suitable output.
What happens to the sample in the flame:
In a typical flame photometric experiment, a solution containing the relevant
substance to be analyzed is aspirated into the burner and dispersed into the flame
as a fine spray. This process is called nebulisation.
In the flame, the solvent evaporates first, leaving finely divided solid particles
which move to hottest region of the flame where gaseous atoms and ions are
produced. The atoms are excited by absorbing energy available from the flame.
As the excited atoms return to a ground state of lower energy, radiation of
wavelength, characteristic of the element, is emitted. The intensity of the emitted
radiation is then measured, which can be related to the concentration of the
element present, which forms the basis of quantitative analysis. The following
processes occur in the flame:
• Desolvation: The sample containing metal particles is dehydrated by the heat of
the flame and the solvent is evaporated.
• Vapourisation: The heat of the flame vapourises the sample constituents. No
chemical change takes place at this stage.
• Atomisation: At this stage the metal ions that were in the solvent are reduced to
metal atoms. For example, Mg2+ (aq) + 2e Mg (g). By heat of the flame and
action of the reducing gas (fuel), molecules and ions of the sample species are
decomposed and reduced to give atoms.
• Excitation: The atoms at this stage are able to absorb energy from the heat of the
flame. The amount of energy absorbed depends on the electrostatic forces of
attraction between the negatively charged electrons and the positively charged
nucleus. This in turn depends upon the number of protons in the nucleus. As
electrons absorb energy they move to higher energy levels and are in the excited
state.
• Emission of radiation: Electrons in the excited state are very unstable and move
back down to the ground state or a lower energy state. As they do so, they
release the energy in the form of radiation of characteristic wavelength, which is
measured by a detector.
 Some metals this radiation corresponds to wavelengths of light in the visible
region of the electromagnetic spectrum and is observed as a characteristic colour
of the flame.
Advantages:
• High sensitivity of flame photometer for most alkali and alkaline earth metal lie in
ppm and sub-ppm.
• Flame photometry is successful in determining certain transition elements like
copper, iron and manganese.

Reagents:
The reagents required are:
• Sodium acetate (NaOAc) 1.0M: Dissolve 136.08 g of sodium acetate trihydrate in
distilled water and bring the volume to 1 litre. Adjust the pH to about 8.2.
• Ethanol- 95 percent.
• Ammonium acetate (NH4OAc) 1.0M: Dissolve 77.09 g of ammonium acetate in
distilled water and dilute to about 900 ml. Adjust the pH to 7.0 with dilute
ammonium hydroxide or acetic acid as required, and make the volume up to 1
litre.
• Standard solution of NaCl: Dissolve 5.845 g of AR-grade NaCl in 1.0M ammonium
acetate and make the volume up to 1 litre. It will give 100 me/litre of sodium in
stock solution. From this solution take 0, 1, 2, 5, 7.5 and 10 ml and make the
volume up to 100 ml each with the ammonium acetate. It will give 0, 1, 2, 5, 7.5
and 10 me/litre of sodium.
Procedure:
The procedure for determining the CEC is:
• Weigh accurately 5 g of soil, and transfer the sample to a 50-ml centrifuge tube.
• Add 25 ml of 1.0M sodium acetate solution to the tube, insert the stopper and
shake in a mechanical shaker for 5 minutes.
• Centrifuge at 2000 rpm for 5 minutes or until the supernatant liquid is clear.
• Decant the liquid completely and repeat the extraction three more times. Discard
the decants.
• Repeat steps 2–4 with ethanol or isopropyl alcohol until the electrical conductivity
(EC) of the decant reads less than 40 mS/cm (it usually takes 4–5 washings).
• To displace the adsorbed Na, repeat steps 2–4 using the ammonium acetate
solution. Collect the decant in a 100-ml volumetric flask fitted with a funnel and
 filter paper. Make up to volume with ammonium acetate solution.
• To determine the sodium concentration by flame photometry, prepare a series of
Na standard solutions in the range of 0–10 me/litre of Na. Prepare a standard
curve by plotting Na concentration on the x-axis and the flame photometric
readings on the y-axis. Feed an unknown sample extract onto the flame
photometer and take the reading, corresponding to which the concentration of Na
is read from the standard curve. For better results, add lithium chloride (LiCl) in
each standard to yield a final concentration of about 5 me/litre of LiCl.

The ammonium acetate extractable Na that is exchangeable Na in me/100 g soil

=Na conc. of extract in meq/litre(Y)×100× Vol. of extract in ml (100)=Y×10=2Y
Wt. of soil in g (5) 1000 5

This displaced Na is actually a measure of the CEC of the soil. Therefore, the me
Na/100 g soil is actually me exchangeable cations (Ca, Mg, Na and K)/100 g soil.
 6. Organic Carbon
The majority of mineral surface soils range from 1.2 to 3.5% organic carbon. Since
soil organic matter averages about 58% carbon, it follows that soils generally
range from about 2 to 6 % organic matter (% O.M. = %C x 1,724. The factor
1.724= 100/58). There is also a close relationship between carbon and nitrogen in
soils. Most organic matter average about 5% nitrogen so that the N : C ratio is
1:11.6. Therefore by multiplying the soil organic matter percentage by 0.05 an
approximate value for the soil nitrogen, percentage is obtained.
In soil the chief source of some of the nutrients essential for plant growth is
organic matter, such nutrients are N, S and boron is also largely derived from
organic matter.

Principle:
A suitable quantity of the soil is digested with chromic acid and Sulphuric acid
making the use of heat of dilution of Sulphuric acid soil is digested and organic
matter of the soil is oxidized. Excess of chromic acid left over unreduced by the
organic matter of the soil is determined by a titration with standard Ferrous
Ammonium sulphate solution using diphenylamine as indicator.
In this exercise, chromic acid in the presence of excess H2SO4 is to be used as an
oxidizing agent for oxidizable organic matter of the soil. The heat of dilution of
H2SO4 works as a standardized ferrous sulphate solution.

4Cr6+ + 3C 4Cr+++ + 3C4+

2H2Cr2O7 + 3C + 6H2SO4 2Cr2(SO4)3 + 3CO2 + 8H2O
K2Cr2O7 + 4H2SO4 K2SO4 + 2Cr(SO4)3 + H2O+ 2O
2FeSO4 + H2SO4 + O Fe2(SO4)3 + H2O

Apparatus and Reagents:

• 500 ml conical flasks.
• Pipette
• Burette
• Phosphoric acid 85%.
• Sodium fluoride 2%.
• Sulphuric acid 96 % containing 1.25 %Ag2SO4
• Standard 1N K2Cr2O7 – 49.04 g/liter.
• Standard 0.5 N Fe(NH4)2(SO4)2.6H2O 196 g in 800 ml water containing 20 cc
H2SO4 and diluted to1 litre.
• Diphenylamine – 0.5g in 20cc water and add 100 ml conc. H2SO4.

Procedure:
• Weigh 1g soil sample in 500 ml conical flask. Add 10 ml of 1 N K2Cr2O7 and 20 ml
conc. H2SO4 (containing Ag2SO4). Mix thoroughly and allow reaction to proceed
for 30 minutes.
• Dilute the reaction mixture with 200 ml water and 10 H3PO4 add 10 ml of NaF
solution and 2 ml of diphenylamine.
• Titrate the solution with standard FAS to a brilliant green colour. A blank without
soil should be run simultaneously.

Observations & Results:

Weight of sample -1g
Normality of K2Cr2O7 used -1N
Vol. of K2Cr2O7 - 10 ml
Normality of FAS - 0.5N
10 0.003 × 100
𝑂𝑂𝑂𝑂% = (Blank − reading) ×
𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 wt. of soil

Limits:
Low : <0.5%
Medium : 0.5 - 0.75%
High : >0.75%
 7. Mineralizable N
Preparation of soil sample:
The soil samples from definite depth are randomly collected from the field with the
help of screw auger. All the possible technical precautions as prescribed for
standard soil sampling are also taken. Samples are then brought to the
laboratory, air-dried in the shade and grounded by wooden roller, thereafter
sieved through 2 mm stainless steel sieve and stored in polythene bags and used
for chemical assay.

Principle:
A known weight of the soil is mixed with alkaline potassium permanganate
(KMnO4) solution and distilled. The organic matter present in soil is oxidized
by the nascent oxygen, liberated by potassium permanganate, in the
presence of sodium hydroxide and the released ammonia is condensed and
absorbed in known volume of a boric acid with mix indicator to form
ammonium borate, the excess of which is titrated with a standard sulphuric
acid.

Reactions involved:
I.Distillation:
Alkaline
2 KMnO4 + H2O 2 MnO4 + 2KOH + 3O-
Medium (Nascent oxygen)
Oxidation
R.CHNH2COOH + O- R.CO.COOH + NH3
Organic- N fraction (Ammonia)
Distillation
NH3 + H2O NH4OH
Absorption
3NH4OH + H3BO3 (NH4)3BO3 + 3H2O
(Green colour)
II.Titration
2(NH4)3BO3 + 3H2SO4 2(NH4)3SO4 + 2 H3BO3
(Pink colour and original)
Equipment and apparatus:
 1. KEL PLUS Automatic Nitrogen Estimation System
The said instrument is used for determination of available nitrogen in soil. It
consists of the following:
• Automatic Distillation System (Model Classic DX): It is fully automatic
distillation system with programmable auto run digital features, with automatic
dilution and addition of boric acid, NaOH and KMnO4. Both modes (auto and
manual) are available for distillation reagents addition.
• Refrigerated Water Cooling System for Condenser (Model Kel Freeze): It is
refrigerated water cooling system for distillation and condensing system with
inbuilt compressor and recirculator pump.
2. Electronic balance
3. Burette
4. Conical flask
5. Distilled water

Reagents:
• 0.32% potassium permanganate (KMnO4) solution.
• 2.5 % sodium hydroxide (NaOH).
• 2 % boric acid solution containing 20-25 ml of mixed indicator / liter.
• Mixed indicator: 0.066g methyl red + 0.099g bromocresol green dissolve in 100 ml
of 95 % alcohol.
• 0.02 N sulphuric acid (H2SO4).

Procedure:
• Weigh 5 g of prepared soil sample and transfer it to the digestion tube.
• Load the tube in distillation unit and other sides of hose keep 20 ml of 2% boric
acid with mixed indicator in 250 ml conical flask.
• 25 ml each of potassium permanganate (0.32 %) and sodium hydroxide (2.5 %)
solution is automatically added by distillation unit programme.
• The sample is heated by passing steam at a steady rate and the liberated
ammonia absorbed in 20 ml of 2 % boric acid containing mixed indicator solution
kept in a 250 ml conical flask.
• With the absorption of ammonia, the pinkish colour turns to green.
• Nearly 150 ml of distillate is collected in about 10 minutes.
• The green colour distillate is titrating with 0.02N sulphuric acid and the colour
 changes to original shade (pinkish color).
• Simultaneously, blank sample (without soil) is to be run.
• Note the blank & sample titer reading (ml) and calculate the available nitrogen
in soil.

Calculations:
𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑁𝑁(𝑘𝑘𝑘𝑘 ℎ𝑎𝑎 −1 )
R(Titer reading − Blank reading) × Normalityof acid × Atomic weight of nitrogen ×
weight of one hectare of soil
=
sample wt. (g) × 1000
R × 0.02 × 14 × 2.24 × 106
=
5 × 1000
𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 = R × 125.44

Interpretation of results:
Available N (kg ha-1) Soil rating
< 280 : Low
280-560 : Medium
> 560 : High
8. Inorganic Nitrogen
Inorganic N in soil is present predominantly as NO3--N and NH4+-N. Nitrite is
seldom present in detectable amounts, and its determination is normally
unwarranted except in neutral to alkali soils following the application of NH4 or
NH4-forming fertilizers.
Nitrate is highly soluble in water, and a number of extractants including water
have been used. These include saturated 0.35 percent CaSO4 2H2O solution,
0.03M NH4F, 0.015M H2SO4, 0.01M CaCl2, 0.5M NaHCO3 (pH 8.5), 0.01M
CuSO4, 0.01M CuSO4 containing Ag2SO4, and 2.0M KCl.
Exchangeable NH4 is defined as NH4 that can be extracted at room temperature
with a neutral K-salt solution. Various molarities have been used, such as 0.05M
K2SO4, 0.1M KCl, 1.0M KCl, and 2.0M KCl.
The potential of a soil to mineralize N as measured by N-availability indices (OM,
organic C and even total N) is fairly constant from year to year, making it
unnecessary to conduct this type of determination each year. However, it is still
necessary to take into consideration the initial amount of available N (inorganic N:
NO3-N and/or NH4-N) in the root zone at or near planting time for better prediction
of N fertilizer needs. This type of test must be conducted each year, especially
where there is the possibility of residual inorganic N remaining from a previous
application or fallow period.
The methods for the determination of NO3-N and NH4-N are more diverse than
the methods of extraction. They range from specific ion electrode to colorimetric
techniques, microdiffusion, steam distillation, and flow injection analysis. Steam
distillation is still a preferred method when using 15N. However, for routine
analysis, this guide details the phenol disulphonic acid method for NO3 and the
indophenol blue method for NH4 estimation.

Nitrate by phenol disulphonic acid method

A major difficulty in estimating NO3 in soils by colorimetric methods is obtaining
a clear colourless extract with low contents of organic and inorganic substances,
which interfere with the colorimetric method. In arid and salt-affected soils,
chloride is the major anion that interferes with colour development of the phenol
disulphonic acid method. Therefore, if the chloride concentration is more than
15 µg/g soil, it should be removed before analysis by the use of Ag2SO4 to
precipitate chloride as AgCl. The Ag2SO4 is added to the extract or to the reagent
 used for extraction, and the AgCl is removed by filtration or centrifugation after
precipitation of the excess Ag2SO4 by a basic reagent such as Ca(OH)2 or
MgCO3.
It is necessary to remove the excess Ag+ before analysis of the extract because it
also interferes with the phenol disulphonic acid method of determining NO3.

Equipments and apparatus:

The apparatus required using the method consists of:
• a reciprocating shaker;
• a heavy-duty hot plate;
• a spectrophotometer;
• a dispenser;
• an Erlenmeyer flask;
• some beakers;
• a glass rod
Instrument:
Spectrophotometer
Spectrophotometry is a method to measure how much a chemical substance
absorbs light by measuring the intensity of light as a beam of light passes
through sample solution. The basic principle is that each compound absorbs or
transmits light over a certain range of wavelength. This measurement can also be
used to measure the amount of a known chemical substance.
A spectrophotometer is an instrument that measures the amount of photons (the
intensity of light) absorbed after it passes through sample solution. With the
spectrophotometer, the amount of a known chemical substance (concentrations)
can also be determined by measuring the intensity of light detected. Depending
on the range of wavelength of light source, it can be classified into two different
types:
• UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm)
and visible range (400 - 700 nm) of electromagnetic radiation spectrum.
• IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
The relationship of light absorbed by the atom in ground state and their
concentration in the solution is defined in the fundamental laws of light
absorptions.
Lambert’s Law: The portion of light absorption by a transparent medium is
independent of the intensity of the incidence light and each successive unit
thickness of the medium absorbs an equal fraction of the light passing through it.
Beer’s Law: Light absorption is proportional to the number of absorbing atoms in
the sample. The combined Beer - Lambert law may be given as:

It = I0 − (abc)

I0
𝑙𝑙𝑙𝑙𝑙𝑙10 = abc = absorbance
It

Where, Io= incident radiation power

It= transmitted radiation power

a= absorption coefficient

b= length of absorption path

c= concentration of absorbing atoms
i.e. the absorbance is proportional to the concentration of the elements for a
given absorption path length at any given wave length.
Components-
The instrument operates by passing a beam of light through a sample and
measuring wavelength of light reaching a detector. The wavelength gives valuable
information about the chemical structure and the intensity is related to the number
of molecules, means quantity or concentration.
All UV-visible spectrophotometer consists of following parts:
Source of radiations-
Commonly used sources for UV region is hydrogen or deuterium discharge lamp
and for visible region tungsten filament is used.
Monochromator-
The light source is a monochromator; the light is split into two equal intensity
beams by a half mirrored device before it reaches the sample. One beam, the
sample beam, passes through a small transparent container (cuvette) containing a
solution of the compound being studied in a transparent solvent. The other beam,
the reference, passes through an identical cuvette containing only the solvent. The
containers for the sample and reference solution must be transparent to the
radiation which will pass through them. Quartz or fused silica cuvettes are
 required for UV-visible spectrophotometer. This kind of arrangement of having
light split up in two beams is known as double beam instrument.
Detector-
The main function is to continuously measure the intensity ratio of the beams
transmitted through sample cell and reference cell. Detector is generally a
photomultiplier tube although in some photodiodes is used.
Recorder-
It automatically records the absorption of light at each wavelength as graph of
absorbance vs. Wavelength.

Reagents:
• Phenol disulphonic acid (phenol 2,4-disulphonic acid): Transfer 70 ml of pure
liquid phenol (carbolic acid) to an 800-ml Kjeldahl flask. Add 450 ml of
concentrated H2SO4 while shaking. Add 225 ml of fuming H2SO4 (13–15 percent
SO3). Mix well. Place the Kjeldahl flask (loosely stoppered) in a beaker containing
boiling water and heat for 2 hours. Store the resulting phenol disulphonic acid
[C6H3OH(HSO3)2] solution in a glass-stoppered bottle.
• Dilute ammonium hydroxide solution (about 7.5M NH4OH): Mix one part NH4OH
(specific gravity 0.90) with one part H2O.
• Copper sulphate solution (0.5M): Dissolve 125 g of CuSO4.5H2O in 1 litre of
distilled water.
• Silver sulphate solution (0.6 percent): Dissolve 6.0 g of Ag2SO4 in 1 litre of distilled
water. Heat or shake well until all salt is dissolved.
• Nitrate-extracting solution: Mix 200 ml of 0.5M copper sulphate solution and 1 litre
of 0.6 percent silver sulphate solution and dilute to 10 litres with water. Mix well.
• Standard nitrate solution (100 µg NO3-N/ml, stock solution): Dissolve 0.7221 g of
KNO3 (oven dried at 105 °C) in water and dilute to 1 litre. Mix thoroughly.
• Standard nitrate solution (10 µg NO3-N/ml, working solution): Dilute 100 ml of 100
µg NO3-N/ml stock solution to 1 litre with water. Mix well.
• Calcium hydroxide: AR-grade powder (free of NO3).
• Magnesium carbonate: AR-grade powder (free of NO3).

Procedure:
• Place about 5 g of soil in an Erlenmeyer flask.
• Add 25 ml of nitrate-extracting solution.
• Shake contents for 10 minutes.
• Add about 0.2 g of Ca(OH)2 and shake for 5 minutes.
• Add about 0.5 g of MgCO3 and shake for 10–15 minutes.
• Allow to settle for a few minutes.
• Filter through No. 42 filter paper.
• Pipette 10 ml of clear filtrate into a 100-ml beaker. Evaporate to dryness on a hot
plate at low heat in a fume hood (free of HNO3 fumes). Do not continue heating
beyond dryness.
• When completely dry, cool residue, add 2 ml of phenol disulphonic acid rapidly
(from a burette having the tip cut off), covering the residue quickly. Rotate the
beaker so that the reagent comes into contact with all residual salt. Allow to stand
for 10–15 minutes.
• Add 16.5 ml of cold water. Rotate the beaker to dissolve residue (or stir with a
glass rod until all residue is in solution).
• Once the beaker is cool, add 15 ml of dilute NH4OH slowly until the solution is
distinctly alkaline as indicated by the development of a stable yellow colour.
• Add 16.5 ml of water (volume becomes 50 ml). Mix thoroughly.
• Read the concentration of NO3-N at 415 nm, using the standard curve.
• Preparation of standard curve: Place 0, 2, 5, 8, and 10 ml of the 10 µg NO3/ ml
working solution in separate 100-ml beakers, add 10 ml NO3-extracting solution
and evaporate to dryness. Follow steps 9–13, using these standard solutions with
0, 0.4, 1.0, 1.6 and 2.0 µg NO3-N/ml. Prepare a standard curve to be used for
estimation of NO3 in the sample.
Calculation:

µ𝑔𝑔
𝑁𝑁𝑂𝑂3− − 𝑁𝑁 𝑖𝑖𝑖𝑖 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠. �
�
𝑚𝑚
vol. after colour develop(ml) Vol. of extracting soln. (ml)
= ×
vol. evaporated(ml) wt. of oven − dried soil(g)
 9. Ammonium by indophenol blue method
Phenol reacts with NH4 in the presence of an oxidizing agent such as hypochlorite
to form a coloured complex in alkaline condition. The addition of sodium
nitroprusside as a catalyst in the reaction between phenol and NH4 increases the
sensitivity of the method considerably. The addition of EDTA is necessary in
order to complex divalent and trivalent cations present in the extract. Otherwise,
it forms precipitate at a pH of 11.4–12 used for colour development, and this
turbidity would interfere with formation of the phenol–NH4 complex.
The first step is the extraction of exchangeable ammonium.

Apparatus:
• The apparatus required consists of:
• an Erlenmeyer flask;
• a volumetric flask;
• a shaker;
• a spectrophotometer
Reagents:
• Potassium chloride (KCl) solution, 2M: Dissolve 150 g of AR-grade KCl in 1 litre of
distilled water.
• Standard ammonium (NH4+) solution: Dissolve 0.4717 g of ammonium sulphate
(NH4)2SO4 in water, and dilute to a volume of 1 litre. If pure dry (NH4)2SO4 is used,
the solution contains 100 μg of NH4-N/ml. Store the solution in a refrigerator.
Immediately before use, dilute 4 ml of this stock NH4+ solution to 200 ml. The
resulting working solution contains 2 μg of NH4-N/ml. Accordingly, for the standard
curve, prepare various concentrations of standard solution.
• Phenol-nitroprusside reagent: Dissolve 7 g of phenol and 34 mg of sodium
nitroprusside [disodium pentacyanonitrosylferrate, Na2Fe(CN)5NO.2H2O] in 80 ml
of NH4+-free water and dilute to 100 ml. Mix well, and store in a dark-coloured
bottle in the refrigerator.
• Buffered hypochlorite reagent: Dissolve 1.480 g of sodium hydroxide (NaOH) in
70 ml of NH4+-free water, add 4.98 g of sodium monohydrogen phosphate
(Na2HPO4) and 20 ml of sodium hypochlorite (NaOCl) solution (5–5.25 percent
NaOCl). Use less or more hypochlorite solution if the concentration is higher or
lower, respectively, than that indicated above. Check the pH to ensure a value
between 11.4 and 12.2. Add a small amount of additional NaOH if required to
 raise the pH. Dilute to a final volume of 100 ml.
• EDTA reagent: Dissolve 6 g of EDTA disodium salt in 80 ml of deionized water,
adjust to pH 7, mix well, and dilute to a final volume of 100 ml.

Procedure:
Extraction:
• Place 10 g of soil in a 250-ml wide-mouth Erlenmeyer flask and add 100 ml of 2M
KCl.
• Insert stopper, and shake the flask on a mechanical shaker for 1 hour.
• Allow the soil–KCl suspension to settle (about 30 minutes) until the supernatant is
clear.
• If the KCl extract cannot be analysed within 24 hours, then filter the soil–KCl
suspension (No. 42 filter paper) and store in the refrigerator.
• Aliquots from this extract are used for the NH4 estimation.
Estimation:
• Pipette an aliquot (not more than 5 ml) of the filtered 2M KCl extract containing
between 0.5 and 12 µg of NH4-N into a 25-ml volumetric flask. Aliquots of ≤ 3 ml
normally contain sufficient NH4-N for quantification.
• Add 1 ml of the EDTA reagent, and mix the contents of the flask.
• Allow the contents to stand for 1 minute, then add 2 ml of the phenol nitroprusside
reagent, followed by 4 ml of the buffered hypochlorite reagent, and immediately
dilute the flask to volume (25 ml) with NH4+-free water, and mix well.
• Place the flask in a water-bath maintained at 40 °C for 30 minutes.
• Remove the flask from the bath, cool to room temperature, and determine the
absorbance of the coloured complex at a wavelength of 636 nm against a reagent
blank solution.
• Determine the NH4-N concentration of the sample by reference to a calibration
curve plotted from the results obtained with 25-ml standard samples containing 0,
2, 4, 6, 8, 10, and 12 µg of NH4-N/ml.
• To prepare this curve, add an appropriate amount of 2M KCl solution (same
volume as that used for aliquots of soil extract, i.e. about 5 ml) to a series of 25-ml
volumetric flasks. Add 0, 1, 2, 3, 4, 5 and 6 ml of the 2 µg NH4-N/ml solution to the
flasks, and measure the intensity of blue colour developed with these standards
by the procedure described above for the analysis of unknown extracts.
 Calculation:
The calculation is (NH4-N in the sample as noted from the standard curve =A
[µg/ml]):
A × 100(total vol. of extract) 1
μg of NH4 − N in 1g soil = ×
5(vol. of extract estimated) 10(wt. of soil)

= 2A
where:
• weight of the soil taken for estimation = 10 g;
• total volume of extract = 100 ml;
• volume of extract taken for estimation = 5 ml.
 10. Available Phosphorus
The phosphorus is an essential plant nutrient and it occurs in many different
forms. Therefore, a reliable procedure for measuring the amount both in soil as
well as in plant is needed. There are many methods available for the
determination, however, colorimetric measurement is presented here:

Principle:
Phosphorus is extracted from the soil with 0.5 m NaHCO3 at a nearly constant pH
of 8.5. The phosphate ion in solution treated with ascorbic acid in an acidic
medium provides a blue colour complex. Measurement of the quantitative
determination of phosphorous in soil.

Reagents:
• 0.5 M Sodium bicarbonate (NaHCO3) solution: Dissolve 42 g of NaHCO3 in
distilled water to get one litre solution and adjust the pH of the solution to 8.5 by
small quantity of NaOH.
• Activated Charcoal: Darco G-60 (P- Free)
• 5 N Sulphuric acid (H2SO4) Solution: Add 141 ml of con. H2SO4 to 800 ml of
distilled water. Cool the solution and dilute to one litre with distilled water.
• Reagent A:
• Dissolve 12.00 g of ammonium molybdate in 250 ml of distilled water.
• Dissolve 0.2908 g of potassium antimony tartrate (KSbO.C4H4O6) in 100 ml
distilled water.
• Above both solution mix thoroughly and made one litre in volumetric flask
with the help of distilled water.
• Add these dissolved reagents to one litre of 5N H2SO4.
• Ascorbic acid working solution (Reagent B): Dissolve 1.056 g of ascorbic acid in
200 ml of reagent A and mix. This ascorbic acid (reagent B) should be prepared
as required because it does not keep more than 24 hours.
• Standard phosphate solution: Weigh 0.4393 g of potassium dihydrogen phosphate
(KH2PO4 ) into one litre volumetric flask. Add 500 ml of distilled water and shake
the contents until the salt dissolves. Dilute the solution to one litre with distilled
water to get 100 ppm P solution. Dilute 20 ml of 100 ppm P solution to one litre to
get form- working solution of 2 ppm.
 Preparation of standard curve:
• Take different concentration of P (0, 1, 2, 3, 4, 5, etc ml of 2 ppm standard P
Solution) in 25 ml volumetric flasks.
• Add 5 ml of the 0.5M NaHCO3 extracting solution to each flask, and acidify with
5N H2SO4 drop by drop.
• Add about 10 ml distilled water and 4 ml of reagent ‘B’, then shake the solution.
• Make the volume 25 ml by distilled water.
• The intensity of blue colour is read on spectrophotometer at 660 nm wavelengths
after 10 minutes.
• Plot the curve by taking P concentration on X axis and colorimeter reading on Y
axis. Repeat the process till you get straight line relationship.
• Calculate the factor i.e. 1 colorimeter reading is equal to how much ppm of
phosphorus.

Procedure:
• Take 2.5 g of soil sample in 150 ml conical flask and 0.5 g Darco G-60 activated
charcoal.
• Then add 50 ml of 0.5 M NaHCO3 solution and shake the solution for 30 minute in
a shaker. Similar processes run for a blank without soil.
• Filter the suspension through the Whatman no. 40 paper.
• Take 5 ml aliquot of the extract in a 25 ml volumetric flask, and acidify with 5N
H2SO4.
• Add small quantity of distilled water, and then add 4 ml of reagent B.
• The intensity of blue colour is read on spectrophotometer at 660 nm wavelengths
after 10 minutes.

Observations:
• Weight of soil sample : 2.5 g
• Volume of extractant used : 50 ml
• Volume of filtrate used : 5 ml
• Absorbency :R
• Absorbency from standard curve : A
• Concentration of P for absorbency A : B ppm
Calculation:
R × F × 50 × 2.24
𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑃𝑃 (𝑘𝑘𝑘𝑘 ℎ𝑎𝑎 −1 ) =
5 × 2.5
𝑊𝑊ℎ𝑒𝑒𝑒𝑒𝑒𝑒 𝐹𝐹 (𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓) = B⁄A

Limits of available P in soil:

Very low : Less than 5 P kg ha-1
Low : 5-10 P kg ha-1
Medium : 10-20 P kg ha-1
High : 20-40 P kg ha-1
Very high : More than 40 P kg ha-1
 11. Available Potassium
The available potassium i.e. exchangeable and water soluble potassium is
determined by extracting soil with neutral normal ammonium acetate solution. The
estimation of potassium is carried out by flame photometer.
Principle:
The principle underlying this is that a large number of elements when excited in a
flame, emit radiation of characteristic wave length. The excitation cause one of the
outer electrons of neutral atoms to move to an outer orbit of higher energy level or
the atoms may be excited sufficiently to lose an electron completely from the
attractive force of the nucleus where excited atom return to lower energy level,
light at characteristic wave length is emitted. Excited atoms or ions give line
radiation at very definite wave length and thus K gives at 404.4 and 767 mµ. The
flame photometer employees a relatively low temperature excitation and
measures with a photocell the emission intensity which is proportional and to
concentration in selected wave length (767 mµ) and for this red filter is used.

Apparatus and reagents:

• Flame photometer with red filter,
• Pipette, volumetric flasks and conical flask (100 ml).

Reagent:
• Neutral Normal Ammonium Acetate: Add 58 ml of glacial acetic acid to about 600
ml H2O and then add 70 ml of concentrated ammonia (sp. gr 0.90). Dilute the
solution to one litre. Then adjust pH of solution at 7.0 with the help of ammonia or
acetic Acid or this can be prepared by dissolving ammo. Acetate (CH3COONH4)
(77.08 eq.wt.) directly in H2O and volume to be made one litre and then adjust the
pH 7.0.
• Standard Potassium Solution: Dissolve 1.9066 g of dried KCl (AR) in distilled
water and dilute to one litre. This is 1000 mg kg-1 K solution. 100 ml of this
solution diluted to 1 lit. to make 100 ppm K solution.

Preparation of the standard curve:

• Take 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and
• 10 ml of 100 mg kg-1 K solution in different 25 ml volumetric flasks. Make up the
volume with 1N NH4OAc Soln. Adjust the flame photometer reading at zero with
 blank (zero K) solution and at 100 for 40 mg kg-1 K solution. Take the flame
photometer readings for every dilution. Plot the standard curve on graph paper by
taking K concentration on X-axis and flame photometer reading a y-axis. This will
give a factor (F) of 1 flame photometer reading = 0.4 mg kg-1 K.

Procedure:
Take 5g soil in 100 ml conical flask and add 25 ml of 1N NH4OAc Soln. Shake the
content for 5 minutes and then filter through Whatman No.1 filter paper.
Potassium extract is measured by flame photometer after calibration.

Calculation:
R × F × 100 × 20 × 1.121
𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝐾𝐾 (𝑘𝑘𝑘𝑘 ℎ𝑎𝑎−1 ) =
5 × 1000
= R × F × 11.217
Limits of available K in soil:
Very low : Less than 200 K kg ha-1
Low : 200 – 250 K kg ha-1
Medium : 250 – 400 K kg ha-1
High : 400 – 600 K kg ha-1
Very high : More than 600 k kg ha-1

Precaution:
• These should not be any turbidity or suspended particles in extract, it will choke
the capillary feeding tube.
• The gas and air pressure should be constant.
• If sample reading goes beyond 100 then dilute the extract.
 12. Available Sulphur
Besides some amount in the soil solution, available sulphur in mineral soils
occurs mainly as adsorbed SO4-2 ions. Phosphate ions (as monocalcium
phosphate) are generally preferred for replacement of the adsorbed SO4-2 ions.
The extraction is also carried out using CaCl2 solution. However, the former is
considered to be better for more efficient replacement of SO4-2 ions. Use of Ca
salts have a distinct advantage over and leads to easy filtration SO4-2 in the
extract can be estimated turbid metrically using a colorimeter/spectrophotometer.
A major problem arises when the amount of extracted sulphur is too low to be
measured precisely.; To overcome this problem, addition of seed solution of
known S concentration is added to the extract to raise concentration to easily
detectable level. Sulphur in the extract can also be estimated by a colorimetric
method using barium chromate, but the turbidimetric method given below is
mainly used in the soil testing laboratories.
Instruments:
• Colorimeter or spectrophotometer or auto analyzer.
• Mechanical shaker
Reagents:
• Mono-calcium phosphate extracting solution (500 mg P L-1): Dissolve 2.035 g of
Ca(H2PO4)2.H2O per litre.
• Gum acacia-acetic acid solution: Dissolve 5 g of chemically pure gum acacia
powder in 500 mL of hot water and filtered in hot condition through Whatman No.
42 filter paper. Cool and dilute to one litre with dilute acetic acid.
• Barium chloride: Pass AR grade BaCl2 salt through 1 mm sieve and store for use.
• Standard stock solution (2000 mg S L-1): Dissolve 1.089 g of oven dried AR grade
potassium sulphate per 100 mL.
• Working standard solution (10 mg S L-1): Measure exactly 2.5 mL of the stock
solution and dilute to 500 mL.
• Barium sulphate seed suspension: Dissolve18 g of AR grade BaCl2 in 44 mL of
hot water and add 0.5 mL of the standard stock solution (given above). Heat the
contents to boiling and then cool quickly. Add 4 mL of gum acacia-acetic acid
solution to it. Prepare a fresh seed suspension for each estimation every day.
• Dilute nitric acid (approx 25%): Dilute 250 mL of AR grade conc. HNO3 to one
litre.
• Acetic-phosphoric acid: Mix 900 mL of AR grade glacial acetic acid with 300 mL
 of H3PO4 (AR grade).
Procedure:
• Weight 20 g of soil sample in a 250 mL conical flask.
• Add 100 mL of the monocalcium phosphate extracting solution (500 mg P L-1) and
shake for one hour. Filter through Whatman No. 42 filter Paper.
• Measure 10 mL of the clear filtrate into a 25 mL volumetric flask.
• Add 2.5 mL of 25% HNO3 and 2mL of acetic-phosphoric acid. Dilute to about 22
mL, stopper the flask and shake well.
• Shake the BaSO4 seed suspension and then add 0.5 mL of it and 0.2 g of BaCl2
crystals. Stopper the flask and invert three times and keep.
• After 10 minutes, invert 10 times and keep. After another 5 minutes, invert 5
times.
• Allow to stand for 15 minutes and then add 1mL of gum acacia-acetic acid
solution.
• Make up the volume, invert three times and keep aside for 90 minutes.
• Invert 10 times and measure the colour intensity at 440 nm (blue filter).
• Run a blank side by side.
Preparation of standard curve for S:
• Place 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 mL portions of the working standard
solution (10 mg S L-1) into a series of 25 mL volumetric flasks to obtain 25, 50,
75, 100, 125 and 150 µg S.
• Proceed to develop turbidity as described above for sample aliquots.
• Read the colour intensity and prepare the curve by plotting readings against
sulphur concentration (In µg in the final volume of 25 mL)
Calculation:
R × 100
𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 𝑆𝑆 𝑖𝑖𝑖𝑖 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 (𝑚𝑚𝑚𝑚𝑘𝑘𝑘𝑘−1 ) =
10 × 20

Where, r stands for the quantity of S in mg as obtained on X-axis against a

reading.
13. Micronutrients - available Zn, Cu, Fe, Mn, B and Mo
All atoms can absorb light at certain discrete wavelengths corresponding to the
energy requirement of the particular atom. When at ground state the atom
absorbs light it is transformed into the excited state. It is the same atom
containing more energy. This energy is measured in relation to the ground state
and a particular excited state say for example in case of Na may be 2.2 eV
(electron volts) above the ground state.
Each transition between different electronic energy states is characterized by a
different energy and by a different wavelength. These wavelengths are sharply
defined and when a range of wavelengths is surveyed, each wavelength shows
as a sharp energy maximum (a spectronic line). These characteristic lines
distinguish atomic spectra. The lines, which originate in the ground state of atom,
are most often of interest in atomic absorption spectroscopy. These are called the
resonance lines. The atomic spectrum, characteristic of each element, then
comprises a number of discrete lines, some of which are resonance lines. Most of
the other lines arise from excited states rather than the ground state. The lines of
excited states are not useful generally in atomic absorption analysis as most of
the atoms in a practical atomizer are found in the ground state.
The relationship of light absorbed by the atom in ground state and their
concentration in the solution is defined in the fundamental laws of light
absorptions.
Lambert’s Law: The portion of light absorption by a transparent medium is
independent of the intensity of the incidence light and each successive unit
thickness of the medium absorbs an equal fraction of the light passing through it.
Beer’s Law: Light absorption is proportional to the number of absorbing atoms in
the sample. The combined Beer - Lambert law may be given as:

It = I0 − (abc)
I0
𝑙𝑙𝑙𝑙𝑙𝑙10 = abc = absorbance
It
Where, Io= incident radiation power
It= transmitted radiation power
a= absorption coefficient
b= length of absorption path
c= concentration of absorbing atoms
i.e. the absorbance is proportional to the concentration of the elements for a
given absorption path length at any given wave length.
In principle, it might be possible to calculate the concentration directly from
the above equation. In practice, however, the a and b are constants hence
the variation of results is directly related the concentration of atoms. For
analysis, the absorbance of different concentration of standard solution is
first measured with the help of atomic absorption spectrophotometer and
then the results of unknown samples are compared with the standards and
thus concentration of unknown sample is calculated.
Atomic absorption spectrophotometer:
Atomic absorption spectrophotometer is based on the principle that when
atomic vapours of an element are irradiated by the radiation of a
characteristic wavelength (i.e. the light from a source whose emission lines
are those of the element in question), they absorb in direct proportion to the
concentration of the element being determined.
Instrument features:
A wide range of atomic absorption spectrophotometer is available today, all of
them have the basic features in common and consist of the following
components:
(a) A Light source:
A Light source emits the spectrum of the element to be determined. The most
widely used light source is hollow cathode lamp which is designed and operated
in such a way that the lines to be measured are sharp, of stable intensity and
free from background.
(b) Atomizer-Burner assembly:
A means of producing atomic vapours of the element to be analyzed. The
solution to be analyzed is drawn by capillary and converted into stream of
compressed air to a fine spray which after condensation of larger droplets is
mixed with the fuel gas acetylene and burnt in a long flame (at 2100-2400oC) in a
stainless steel burner.
(c) A Monochromator:
It isolates the absorbing resonance lines from other non-absorbing lines. When
the light coming from the HCL, after traversing the flame, enters the
monochromator which is already set at the wavelength of the resonance lines of
the desired element, the monochromator performs its function.
(d) A Detector:
It measures the magnitude of absorption of the characteristic radiation.
(e) A Photomultiplier Tube:
It amplifies the absorption signal and converts the light radiation into electrical
energy.
(f) A readout system:
It measures the absorbance in volts. It is normally a strip chart recorder, a digital
display, a meter or printer. The presently available AAS have features like
automatic calibration with one or more standards, automatic curve corrections,
automatic and foolproof gas switching and calculation of average and standard
deviations in repetitive runs.

Collection and preparation of soil:

To avoid contamination, soil samples are to be collected in plastic tub, using rust
free instrument or wood and kept in polythene lined cloth bags. Samples are
prepared with the help of wooden mortar and pestle and sieved through 2mm
nylon screen/mosquito net cloth or stainless steel sieve.
Soil extraction: DTPA offers the most favourable combination of stability
constants for the simultaneous complexing of Zn, Cu, Fe and Mn, Cd, Co, Ni
and Pb. Buffering of extractant in a slightly alkaline pH range (7.3) by
including soluble Ca2+, avoids the dissolution of CaCO3 with the release of
occluded micronutrients due to CO2 partial pressure of approximately 10
times that in atmosphere, as the soil contains slightly higher CO2 levels than
found in the atmosphere.

(a) Extracting solution: (0.005 M DTPA) Dissolve 1.9679g of DTPA

(Diethylene tri amine penta acetic acid) + 13.3 ml TEA (Triethanol amine) +
1.47g CaCl2 .2H2O in 200 ml distilled water, dilute to 900 ml, adjust pH 7.3
with 6N HCl while stirring and then make upto 1 liter and mix thoroughly.
(b) Apparatus required: Shaker (Horizontal or Rotatory), iodine value flasks
(100 ml capacity) or conical flasks with glass stoppers, funnels, filter paper
whatman No.1, plastic storage bottles and Atomic absorption
spectrophotometer.
(c) Stock Standard Solutions: The standard solutions of different micronutrients
should preferably be prepared by using their wires. Dissolve 1g wire in a minimum
volume of 1:1 nitric acid and dilute to 1000ml with distilled water to obtain 1000
µg/ml solution of micronutrient, or take salts of metals as follows:
Zn- 4.398g l-1 ZnSO4.7H2O
Cu- 3.929g l-1 CuSO4.5H2O
Fe- 4.977g l-1 FeSO4.7H2O
Mn- 3.598g l-1 MnSO4.H2O
The prepared standards are also available in the market. Out of these standards,
prepare working solution of 50 ppm. Then a series of standard solution of 0.5, 1.0,
1.5, 2.0 and 2.5 ppm may be prepared for each metal.
(d) Soil analysis: Weigh 12.5g soil sample in 100 ml iodine value flasks. Add
25 ml DTPA solution. Shake this mixture for 2 hours on shaker at 70 to 80
oscillations per minute, filter through acid washed distilled water rinsed
whatman No.1 filter paper and collect the filtrate in plastic bottles. Determine
the content of micronutrients on atomic absorption spectrophotometer.
Factors:
For soil multiply the concentration read on AAS computer sheet by “2”.

Availability Micronutrients (μg/g soil)

Zn Cu Fe Mn
Very low 0–0.5 0–0.1 0–2 0–0.5
Low 0.5–1 0.1–0.3 2–4 0.5–
1.2
Medium 1–3 0.3–0.8 4–6 1.2–
3.5
High 3–5 0.8–3 6–10 3.5–6
Very high >5 >3 > 10 >6